We propose to extend and combine a series of analytical tools for general use in the interpretation and analysis of UV-visible spectra of typical chemical and biochemical systems. The widespread use of digitizing, fast- scanning spectrophotometers by investigators studying a wide range of problems, has created the need for the development and availability of these techniques. This development will be brought about through the study of a series of progressively more complicated heme (iron porphyrin)-ligand equilibria. The first system to be studied is the titration of mesotetraphenylporphyrinatoiron(II), Fe(II)TPP, with 1,2-dimethylimidazole, 1,2-DMI. Since this base only binds once-at moderate concentrations-its titration consist of two components; Fe(II)TPP and Fe(II)TPP(1,2-DMI). The two corresponding pure UV-vis spectra are easily obtainable and so standard methodologies will be used to reproduce the binding constant. The spectral curves of this titration will then be further treated as follows: 1) Factor Analytical decomposition (FA), and 2) Fitting to a series of Gaussian/Lorentzian functions (GLF). The goal behind using these analysis is to determine the accuracy with which each can reproduce; a) the binding constant, and b) the individual pure spectra. In addition to the statistical methods of each of these two analysis, each of these two techniques uses nonlinear least-squares curve fitting procedures. The Marquardt algorithm is a very efficient method of doing this, but may miss global minima if the initial guesses are off. Methods for improved searching of parameter space will be explored. Following work on the two component system the FA and GLF techniques will be extended to systems composed of three, four, six and finally nine components. These systems will be composed of various combination of the iron porphyrins Fe(II)TPP and meso-tera(alpha, alpha, alpha, alpha-o- pivalamidoporphyrinatoiron(II), iron(II)(TpivPP), "picket fence" heme, and the bases 1-Methylimidazole, 1,2-dimethylimidazole, 1,5- dicyclohexylimidazole, pyridine and 4-phenylpyridine. Since several of the pure spectra and binding constants are known, or estimates of them are available, the development and accuracy of the proposed analyses will be tested, and critically evaluated, at each step. The ligands to be used are all commercially available as are the porphyrins with the exception of one, "picket fence" porphyrin, whose synthesis and purification is a series of 2 high yield steps: 1) condensation of meso-tera-alpha, alpha, alpha, alpha-o-aminopnenylporphyrin which is isolated form the commercially available mixture of atropisomers via column chromatography. 2) Insertion of iron to this porphyrin by adding FeBr to this porphyrin in refluxing THF/CHCl3. Software will be developed for both mainframe and microcomputers. The tentative timetable is to set up the lab and characterize the two and three component systems in the first year. Extensions to the more complicated systems will take up the second year and most of the third. Following years will see applications of the software to different systems of biochemical interest.